The complement system helps or “complements” the ability of antibodies and phagocytic cells to clear pathogens from an organism. It is part of the immune system called the innate immune system that is not adaptable and does not change over the course of an individual's lifetime. However, it can be recruited and brought into action by the adaptive immune system. The complement system consists of a number of enzymes, proenzymes, and other proteins which form the principal effector mechanism of immunity in extracellular body fluids (especially blood plasma). When stimulated by one of several triggers, proteases in the system cleave specific proteins to release cytokines and initiate an amplifying cascade of further cleavages. The end-result of this activation cascade is massive amplification of the response and activation of the cell-killing membrane attack complex. Over 25 proteins and protein fragments make up the complement system, including serum proteins, serosal proteins, and cell membrane receptors. Three biochemical pathways activate the complement system: the classical complement pathway, the alternative complement pathway, and the lectin pathway.
Common clinical characteristics of the thrombotic microangiopathies (TMA), thrombotic thrombocytopenic purpura (TTP) and atypical hemolytic-uremic syndrome (aHUS), include microvascular platelet adhesion/aggregation/occlusion, thrombocytopenia, and mechanical hemolysis. Organ dysfunction in TTP is usually central nervous system, gastrointestinal or renal. In hemolytic-uremic syndrome (HUS), either shiga-toxin induced (D-HUS) or aHUS, organ dysfunction is predominantly renal. TTP is often associated with a deficiency of functional ADAMTS-13 (mutations or autoantibody-inhibited), the protease responsible for regulating the size of circulating von Willebrand factor (VWF) multimers. D-HUS results from infection of shiga-toxin harboring bacteria; and aHUS is developed from autoantibodies or defects in proteins of the alternative complement pathway (AP).
Endothelial cells (ECs) that line blood vessels produce and secrete into plasma: VWF, ADAMTS-13 and all the components of both the classical complement pathway (CP) and the AP. ECs store and secrete ultra-large (UL) von Willebrand factor (ULVWF) multimers with molecular masses over 10 thousand kDa and lengths up to 100 μm. The cell anchored ULVWF strings are hyper-adhesive to platelets and capable of producing microvascular thrombi before cleavage by ADAMTS-13 into less active smaller VWF forms. There is an accumulation of ULVWF strings on endothelial cell (EC) surfaces under conditions when the ULVWF strings are secreted at increased rates combined with lower amounts of functional ADAMTS-13. Bacterial toxins, inflammatory cytokines, phosphodiesterase inhibitors and calcium ionophore are among the agents that cause increased rates of ULVWF secretion from ECs. aHUS is the result of excessive complement activation or, more commonly, defective regulation of proteins of the AP. The primary effect of uncontrolled AP activity in aHUS is damage to renal endothelium, resulting in renal failure.
Although it has been established that AP regulation is dysfunctional in aHUS, it is unclear what initiates the AP activation. Limited activation of the AP can begin by direct hydrolysis of an intra-molecular bond in C3 to C3-H2O. Subsequent cleavage activation of C3, releasing 9 kDa fragment C3a to form C3b, and further amplification of C3b production depends on the presence of “activating surfaces.” C3b (not intact C3) attaches covalently via an exposed thioester to hydroxyl-containing amino acids (threonine, serine and tyrosine) on activating surfaces. C3b then binds factor B (FB) to produce C3bB. FB in the C3bB complex is cleaved to active Bb by factor D (FD) to produce C3bBb, the AP C3 convertase (with t1/2 of 1-3 min) that is stabilized by factor P (properdin; FP). The Bb in C3bBb on an activating surface cleaves fluid-phase C3 to generate additional surface-bound C3b, a process that rapidly amplifies C3b generation from C3. As the ratio of C3b to Bb increases, C3bBbC3b is formed (as the AP C5 convertase), binds C5 with high affinity, and cleaves C5 to C5b. C5b combines with C6 and C7 to generate C5b67 complexes that insert into cell membranes. If C8 and multiple C9 molecules combine with C5b67 complexes in the cell membrane, then lytic C5b678(9)n terminal complement complexes (TCCs) are formed.
Factor H (FH) and factor I (FI) are fluid-phase negative regulatory proteins of the AP. FH can displace Bb from C3bBb and C3bBbC3b complexes and enables FI to cleave and inactivate C3b. Heterozygous mutations of the CFH gene or autoantibody-mediated inhibition of FH are prominent causes of aHUS. aHUS is also associated with heterozygous loss-of-function mutations of CFI, and heterozygous gain-of-function mutations in C3 or CFB.
In contrast to the AP, the CP and lectin-activated complement pathway (LP) are initiated by C1 (complex of C1q6, C1r2, C1s2) attachment to antigen-antibody aggregates or mannose/N-acetylglucosamine-binding lectin (MBL)/MBL-associated protein (MASP), respectively. Both the CP and LP lead to cleavage and activation of C4 and C2 to generate C4b2a complexes. Analogous to activated C3b, activated C4b has an exposed thioester capable of binding covalently to surfaces. The C2a protease in C4b2a (the classical/lectin pathway C3 convertase) cleaves C3 into active C3b.
Human ECs of a variety of types (umbilical vein, arterial, lung microvascular, glomerular microvascular) secrete and anchor ULVWF strings in response to many stimuli. In vivo, EC-secreted/anchored ULVWF strings are exposed to all of the complement components in the circulation. After verifying and quantifying human umbilical vein endothelial cell (HUVEC) expression of complement proteins, we initially studied ULVWF strings and the attachment of complement components that were released exclusively from cultured ECs in the absence of other plasma proteins. ULVWF multimers are compressed in WPBs in a spring-like conformation that allows its rapid unfolding to the EC surface after stimulation, without additional application of shear stress or flowing conditions.